Recent studies with the gram-negative bacterium Escherichia coli have indicated that proteins are exported to the periplasmic space and the outer membrane of this organism by a mechanism similar to that proposed for the initial steps of protein secretion in eukaryotic cells. Using E. coli as a model, we wish to understand the various factors that determine protein localization in this organism. We have recently developed a selection procedure which permits us to isolate E. coli mutants defective in the secretion of the maltose binding protein (MBP) into the periplasm. In these mutants, the MBP accumulates in the cytoplasm in its higher m.w. precursor form. Using this same selection procedure, we propose to undertake a detailed genetic and biochemical analysis of the secretion of the MBP. We will define the important structural features of the MBP precursor which are responsible for insuring the export of the MBP to the periplasm, and we will attempt to understand how the cell's secretory apparatus recognizes the MBP as a secreted protein. In particular, we will focus on the amino-terminal "signal sequence" of this protein. In addition, we wish to determine the mode of processing of the MBP to its mature form, and whether or not complete or partial processing of the MBP precursor is prerequisite for translocation of the MBP across the cytoplasmic membrane. FinallY, we will determine if the MBP and other exported proteins in E. coli can be classified on the basis of a competition for specific export sites on the cytoplasmic membrane. These research goals, if achieved, should provide us valuable insight into understanding the early steps in the localization process for membrane and secreted proteins in both prokaryotic and eukaryotic systems.